A new series of choline acetyltransferase (ChA) inhibitors has been discovered. The parent compound, a derivative of Hemicholinium No. 3 (HC-3), is a selective inhibitor of ChA: it is not a potent inhibitor of cholinesterases. Since it is active in vivo as well as in vitro, it provides a unique tool for the study of cholinergic mechanisms in normal and in pathological states in experimental animals. Several analogues of this compound are also being evaluated. A new series of fluorescent hemicholiniums is being investigated. Since the drugs themselves are fluorescent, their distribution, sites of action, and metabolism can be studied using fluorescence microscopy and spectrophotofluorimetry. Thus, the difficulties and danger inherent in the use of radiolabeled compounds are absent. Furthermore, the parent compound is more potent than HC-3 itself. Among the methods to be used are: isolated, purified brain choline acetyltransferase, choline distribution in mouse brain mince, fluorescence microscopy as well as standard pharmacological preparations to evaluate the drugs' actions on the cardiovascular system and the neuromuscular junction. These compounds should provide new ways to study the synthesis of acetylcholine and its role in cortical arousal, the acquisition of conditioned avoidance responses, myasthenia gravis and myasthenic syndrome, epilepsy, and other conditions in which acetylcholine has a known or putative function.